A biomolecule or biological molecule is any molecule that is present in living organisms, including proteins, carbohydrates, lipids, and nucleic acids. With advances in physics, biochemistry and bioinformatics, techniques for analyzing biomolecules and producing comprehensive information on quantitative states, that is, profiles, of biomolecules in biological samples have been developed. However, there is still a need for a novel, efficient method and apparatus due to problems with conventional methods and apparatuses, including difficulty of use, maintenance costs, feasibility, accuracy, sensitivity, testing time and process automation ability.
Techniques for producing a profile of a biomolecule in a biological sample, although not the ultimate object but a means to approach the object, find a wide range of application in various fields including medicine, veterinary science, environmental engineering, food engineering, the agriculture industry and the like.
Profiles of biomolecules including nucleic acid, proteins and other organic substances as constituents of tissues, cell mass, microorganisms, etc. are constructed by means of various methods using physical and chemical properties.
A clinical decision support system is a health information technology system that is designed to analyze the biological significance of biomolecules in biological samples using the profiles, with the aim of performing decision making tasks for physicians and other health professionals for diagnosis and treatment. Clinical decision support systems are largely classified as Case-Based Machine Learning Inference Systems and Expert Systems. In the Case-Based Machine Learning Inference Systems, the clinical information and biological information of known disease-carrying patients, that is, the profiles of biomolecules of patients, are collected, and a disease of interest is inferred or determined from the given clinical and biological information using machine learning on the basis of the collected data. The Expert System is a computer system that is designed to diagnose a disease using rules set forth by medical experts.
With regard to nucleic acids and proteins representative of biomolecules, genetic information is stored in a deoxyribonucleic acid (DNA), which is organized into long structures called chromosomes. About 3 billion nucleotides are included in the human genome. The nucleotide sequences in each chromosome play a critical role in forming the characteristics of individual subjects. Many diseases are based on modifications of the nucleotide sequences in the human genome. Gene codes in genomes belonging to individuals of the same species may differ from one biological individual to another, exhibiting variation in nucleotide sequence, called polymorphism. Causes of polymorphism include the deletion and/or insertion of at least one nucleotide, and the repetition of a certain base sequence. Single nucleotide polymorphism (SNP) is a variation in a single nucleotide that occurs at a specific position in the genome.
Genotypic chemistry analysis methods for many SNPs include PCR-restriction fragment length polymorphism analysis, single-strand conformation polymorphism detection, dideoxy minisequencing, oligonucleotide ligation assays, allele-specific polymerase chain reaction (hereinafter referred to as “AS PCR”) analysis, ligase chain reaction analysis, primer-required nucleotide incorporation assays, and fluorescence energy transfer-based assays (Landegren, U., et al., 1998, Genome Res, 8:769-776; Gut, I. G., 2001, Hum Mutat, 17:475-492; Shi, M. M., 2001, Clin. Chem., 47:164-172). In addition, mass spectrometry (Ross P et al., 2000, BioTechniques, 29:620-629) and oligonucleotide microarray-based analysis (Wang, D. G. et al., 1998, Science, 280:1077-1082) have recently been suggested for direct determination of the mass of short single DNA fragments with accuracy.
Allele-specific hybridization was developed for Affymetrix whole genome SNP array (Komura D., et al., 2006, Genome Res. 2006; 16: 1575-84. 6), Idaho Hi-Res Melting curve analysis system (Graham R., et al., 2005, Clin. Chem. 51: 1295-8), dynamic allele-specific hybridization (DASH) (Prince J. A., et al., 2001, Genome Res. 11: 152-62), and Illumina Golden Gate SNP Genotyping Arrays (Gunderson K. L., et al., 2005, Nat Genet. 37: 549-54), and fluorescence resonance energy transfer (FRET) is used in TaqMan (Holloway J. W., et al., 1999, Hum Mutat. 14: 340-7). Molecular beacon assays were developed and described in Barreiro L. B., et al., 2009, Methods Mol Biol. 578: 255-76. Extension techniques used AS PCR are found in Illumina Infinium bead array (Oliphant A., et al., 2002, Biotechniques. Suppl: 56-8, 60-1), Beckman GenomeLab SNPstream system (Bell P. A., et al., 2002, Biotechniques. 2002; Suppl: 70-2, 74, 76-7), and Sequenom MassARRAY SNP system (Hayes B. J., et al. 2007, Bioinformatics. 2007; 23: 1692-3).
Since such techniques for identifying SNP exhibit relative advantages and disadvantages depending on the purpose, it cannot be said that one technique is better than another. However, a DNA-based microarray technique has attracted great attention because it can simply analyze the existence, quantity or expression pattern of specific genes or gene clusters (Schena et al., 1995, Science, 270:467-470; DeRisi et al., 1996, Nature Genetics 14:457-460).
In each cell, about 50,000-100,000 genes exist, but are selectively used. A significant number of the genes are required for the maintenance of basic cellular functions. These genes are called housekeeping genes (hereinafter referred to as “HKG”). In addition, an endogenous standard expression gene is used as a control in revealing the function of a certain gene, searching for a gene having a specific function, or examining the expression pattern of a gene under given conditions. According to various molecular biological purposes, the quantitative analysis of messenger RNA (hereinafter referred to as “mRNA”) is used to compare expression levels of certain or multiple genes, as implemented by many techniques including reverse transcriptase polymerase chain reaction (hereinafter referred to as “RT-PCR”), quantitative real time PCR (hereinafter referred to as “qRT-PCR”), serial analysis of gene expression (hereinafter referred to as “SAGE”) or microarray assay.
However, conventional DNA microarrays, which are designed to detect target sequences only through hybridization, are still plagued by the problem of false positives due to cross reaction, and thus require improvement in the reliability of the hybridization signal. In addition, conventional microarrays require very strict post-hybridization washing steps and indispensably require the denaturation of a target sequence into a single strand prior to hybridization. On-chip PCR has recently been developed as a heterogeneous assay system, like conventional microarrays, to detect target genes through hybridization or probe extension, but suffers from the disadvantages of impossibility of real-time detection and difficulty of accurate quantitation.
In addition, protein chips or aptamer chips have been developed on the basis of high-throughput screening techniques to track the interactions and activities of proteins, and to determine their functions (Smith et al., Mol Cell Proteomics, 11-18. 2003; and McCauley et al., Anal Biochem, 319(2), 244-250. 2003). The chip consists of a support surface, such as a glass slide, nitrocellulose membrane, bead or microtiter plate, to which an array of capture proteins is bound.
Further, biomolecules are identified by profiling, isolated, and analyzed for their constitution by MALDI-TOF (Matrix Assisted Laser Desorption/Ionization-Time Of Flight). In recent years, protein profiles have been under extensive study using SELDI-TOFMS (Surface-Enhanced Laser Desorption/Ionization Time Of Flight Mass Spectrometry) (Adam et al., Cancer Research, 62, 3609-3614. 2002; Li et al., Clinical Chemistry, 48, 1296-1304. 2002; and Petricoin et al., The Lancet, 359, 572-577. 2002). As a further approach, immuno-PCR (IPCR), in which signals are amplified using a DNA and polymerase (Sano et al., Science 258, 120-122. 1992), has been suggested.
As described above, there have been many developments in such assays in terms of detection sensitivity and multiple analytical potential, but they are in need of the reduction of cost and time, and the improvement of sensitivity and reproducibility.
The present inventor suggested a reverse-SELEX method for producing profiles of proteins (Korean Patent No. 10-0670799), an aptamer-based nucleic acid chip (Korean Patent No. 10-0464225), a method for analyzing biological significance using an aptamer-based nucleic acid chip (Korean Patent No. 10-0923048), and a method for analyzing genetic variation (Korean Patent Application No. 10-2013-0118222), but they are unable to analyze both proteins and nucleic acids simultaneously in biological samples, which can be achieved in the present disclosure.
The comprehensive analysis of biomolecules in biological samples allows for the production of profiles of biomolecules associated with diseases.
Comprehensive research into biomolecules of biological samples may be characterized by analyzing profiles of biomolecules associated with diseases, thereby identifying biomolecules that allows for the diagnosis of diseases or the analysis of therapeutic effects, biomolecules playing an important role in the onset and progression of diseases, biomolecules responsible for susceptibility to diseases, and target molecules for the development of novel drugs.
Leading to the present invention, intensive and thorough research into the analysis of biomolecules, conducted to overcome the problems encountered in the conventional techniques for analyzing biomolecules separately, resulted in the development of a method for analyzing all of the biomolecules responsible for a given biological event at once, in real time with improved efficiency and sensitivity.
Ultimately, the holistic analysis of biomolecules in a biosample with the production of biological profiles thereof can be applied to the diagnosis of diseases, the analysis of therapeutic effects, and the identification of biomolecular principles that play an important role in the onset and progression of disease or which are responsible for susceptibility to disease, and target molecules for the development of novel drugs.